Our laboratory is focused on the manipulation of immune responsiveness for the prevention of undesirable or ineffective immune responsiveness, primarily in autoimmunity. Our approach has been to utilize immunoglobulin fusion proteins delivered via retroviral vectors for tolerance. This technology is based on the tolerogenicity of immunoglobulin carriers, onto which we engineer multiple epitope- containing polypeptides in frame with this IgG scaffold. Data in two experimental autoimmune models (uveitis and EAE) are promising in that significant clinical efficacy has been achieved. That is, when expressed in bone marrow-derived cells or LPS blasts via retroviral infection, the polypeptide-IgG induces significant hyporesponsiveness to both T-cell and B-cell epitopes in model systems, and can both prevent and reverse autoimmune responsiveness in uveitis and EAR Preliminary results in NOD mice as a model for diabetes are encouraging in that either an insulin B chain-IgG or a GAD-IgG construct can be used to transfect B cell blasts and provide clinical protection in NOD mice even after the onset of insulitis! However, it is not clear what the best target antigens/epitopes in this platform technology are for long-term tolerance in diabetes, nor the best delivery cells and vectors our gene therapy for tolerance. In this proposal, we wish to engineer multiple epitopes of GAD and insulin B chains into retroviral fusion protein constructs for effective tolerance induction, as measured by insulitis, as well as immune parameters, such as cytokine responsiveness. To explore mechanisms, we will examine the efficacy of different target cells for gene therapy and the fate of insulin-specific T cells. Finally, we will apply these results to an FIV based non-primate lentivirus vectors for tolerance induction as an important step toward future clinical trials for diabetes.